A plethora of potent human pathogens uses type IV pili (T4Ps) as essential elements of their pathogenic strategy. Common components of the complex nanomachine that assembles and retracts T4Ps could present vulnerabilities to be exploited by researchers to develop vaccines and therapeutics. Critical barriers to progress toward these efforts include the rudimentary understanding we have of the structure and function of T4P biogenesis machines and the lack of knowledge regarding which aspects of these machines are common among all bacteria that produce T4Ps. In this proposal we describe a series of experiments designed to transcend these barriers. We plan to test the hypothesis that an inner membrane protein with homologues, apparent or cryptic, in all T4P systems carries out an essential initial step by recruiting the mature pilin protein to the machine We will characterize this step to the atomic level using a model system. We will also elucidate the function of pilin-like proteins, which play critical roles in pilus dynamics that have until no eluded experimental investigation. Our plan to extend experiments performed in one model system to a second system, separated from the first by eons of evolution, is a novel aspect of this proposal. We will focus our efforts on enteropathogenic Escherichia coli (EPEC), a cause of lethal diarrhea in developing countries that relies on T4P for full virulence. We have over twenty years of experience working on this system and our prior discoveries and cadre of tools portend rapid advances with this model. Our more recent experience studying the T4P system of Clostridium difficile, another lethal pathogen, puts us in a unique position to determine whether our findings in EPEC apply also to this remote relative and therefore have potential universal import. Our cross-disciplinary approach and talented team of investigators, including experts in microbiology, bacterial genetics, protein chemistry, structural biology, and biophysics, further enhance our prospects for progress. The experiments described in this proposal will provide a wealth of information that is essential for our long-term goal of a complete understanding of T4P biogenesis. The knowledge we gain regarding common elements of the process may have practical implications for vaccine development and new therapeutics.